Identification and Characterisation of cis-Regulatory Elements Upstream of the Human Receptor Tyrosine Kinase Gene MERTK

Abstract

Background: MERTK encodes a receptor tyrosine kinase that regulates immune homeostasis via phagocytosis of apoptotic cells and cytokine-mediated immunosuppression. MERTK is highly expressed in the central nervous system (CNS), specifically in myeloid derived innate immune cells and its dysregulation is implicated in CNS pathologies including the autoimmune disease multiple sclerosis (MS).

Objective: While the cell types and tissues that express MERTK have been well described, the genetic elements that define the gene's promoter and regulate specific transcription domains remain unknown. The primary objective of this study was to define and characterise the human MERTK promoter region.

Methods: We cloned and characterized the 5' upstream region of MERTK to identify cis-acting DNA elements that promote gene transcription in luciferase reporter assays. In addition, promoter regions were tested for sensitivity to the anti-inflammatory glucocorticoid dexamethasone.

Results: This study identified identified both proximal and distal-acting DNA elements that promote transcription. The strongest promoter activity was identified in an ∼850 bp region situated 3 kb upstream of the MERTK transcription start site. Serial deletions of this putative enhancer revealed that the entire region is essential for expression activity. Using in silico analysis, we identified several candidate transcription factor binding sites. Despite a well-established upregulation of MERTK in response to anti-inflammatory glucocorticoids, no DNA region within the 5 kb putative promoter was found to directly respond to dexamethasone treatment.

Conclusions: Elucidating the genetic mechanisms that regulate MERTK expression gives insights into gene regulation during homeostasis and disease, providing potential targets for therapeutic modulation of MERTK transcription.

Keywords: MERTK; gene expression regulation; multiple sclerosis; myeloid cells; promoter regions; transcription.

Fig. 1

5’ upstream MERTK sequence contains regions which function as cis-acting promoter elements. Functional activity of generated MERTK promoter amplicons was assessed via luciferase reporter activity. A. Schematic representation showing the design of pGL3 reporters relative to the MERTK promoter region and their functional promoter activity. B. Schematic representation of a putative proximal promoter region relative to the MERTK gene with corresponding CpG islands. Numbers next to each CpG track represent the sum of consecutive CpG doublets for that island. Data obtained from UCSC Genome Browser (GRCh37/h19) C. Sequence alignment of MERTK transcription start site (+1) and the consensus sequence for a typical mammalian promoter initiator site with matching bases highlighted. Consensus symbols; Y = pyrimidine (C or T); N = any base; W = A or T. Luciferase expression was measured 48 hours following transfection in HEK293T cells. Relative luminescence represents ratio of firefly luciferase activity to the co-transfected renilla luciferase control. Data are expressed as means±SEM. Data are representative of 3 independent experiments (except when construct is denoted with a ^#, which is data representative of 1 experiment). Difference in activity of the pGL3 constructs was tested against the empty pGL3 enhancer vector using a paired ratio t test, * P < 0.05.

Fig. 2

Comparison between proximal promoters of MERTK and AXL reveals similar enrichment for SP1 and SP3 binding sites. Partial nucleotide sequences (–300/+1) of 5’ flanking region of human and mouse MERTK and human AXL. A. Pairwise comparison of human and mouse sequences reveals conserved SP1 and SP3 consensus sequences (highlighted red) predicted by JASPAR and experimentally observed as binding sites in mouse sertoli cells [35]. Conserved binding sites predicted by JASPAR only are highlighted in blue. B. SP1 and SP3 binding sites in AXL proximal promoter have been experimentally observed in Rko and HeLa cells [47]. Sequence data obtained from UCSC Genome Browser (GRCh37/hg19 for human and GRCm38/mm10 for mouse).

Fig. 3

MERTK-3 deletion constructs reveal entire 859 bp region is required for observed expression. Functional activity of MERTK-3 deletion constructs was assessed via luciferase reporter activity. A. Schematic representation of MERTK-3, flanking clones MERTK-2 and MERTK-4 and the corresponding region deleted by each MERTK-3 deletion construct (MERTK-3Δ S1–4) with accompanying ENCODE ChIP experimental data and TRANSFAC analysis of transcription factor binding sites. Data obtained from UCSC Genome Browser (GRCh37/hg19), ENCODE and TRANSFAC databases. B. Schematic representation showing the design of MERTK-3 deletion constructs relative to the full MERTK-3 vector and their functional promoter activity. Relative Luminescence represents ratio of firefly luciferase activity to the co-transfected renilla luciferase control. Data are expressed as means±SEM. Data are representative of 3 independent experiments. Data were analysed by a one-way ANOVA with post-hoc comparisons between MERTK-3 and each individual deletion construct. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

MERTK promoter activity is not enhanced following treatment with dexamethasone. Functional activity of MERTK promoter constructs was assessed via luciferase reporter activity following treatment with 100 nM dexamethasone. Each construct was co-transfected with a pCMV6-Xl5 vector encoding the full-length human cDNA for the glucocorticoid receptor NR3C1. The commercially available Pathdetect GRE-luc was used as a positive control for dexamethasone response. Relative Luminescence represents ratio of firefly luciferase activity to the co-transfected renilla luciferase control. Data are representative of one independent experiment and are expressed as means±SD. Data were analysed by a one-way ANOVA with post-hoc comparisons between treated and untreated constructs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.